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Overexpression and enzymatic characterization of Neisseria gonorrhoeae penicillin-binding protein 4.
- Source :
- European Journal of Biochemistry; Jan2004, Vol. 271 Issue 1, p23-32, 10p
- Publication Year :
- 2004
-
Abstract
- The penicillin-binding proteins (PBPs) are ubiquitous bacterial enzymes involved in cell wall biosynthesis, and are the targets of the β-lactam antibiotics. The low molecular mass Neisseria gonorrhoeae PBP 4 (NG PBP 4) is the fourth PBP revealed in the gonococcal genome. NG PBP 4 was cloned, overexpressed, purified, and characterized for β-lactam binding, dd-carboxypeptidase activity, acyl-donor substrate specificity, transpeptidase activity, inhibition by a number of active site directed reagents, and pH profile. NG PBP 4 was efficiently acylated by penicillin (30 000 m<superscript>−1</superscript>·s<superscript>−1</superscript>). Against a set of five α- and ε-substituted l-Lys- d-Ala- d-Ala substrates, NG PBP 4 exhibited wide variation in specificity with a preference for N<superscript>ε</superscript>-acylated substrates, suggesting a possible preference for crosslinked pentapeptide substrates in the cell wall. Substrates with an N<superscript>ε</superscript>-Cbz group demonstrated pronounced substrate inhibition. NG PBP 4 showed 30-fold higher activity against the depsipeptide Lac-ester substrate than against the analogous peptide substrate, an indication that k<subscript>2</subscript> (acylation) is rate determining for carboxypeptidase activity. No transpeptidase activity was apparent in a model transpeptidase reaction. Among a number of active site-directed agents, N-chlorosuccinimide, elastinal, iodoacetamide, iodoacetic acid, and phenylglyoxal gave substantial inhibition, and methyl boronic acid gave modest inhibition. The pH profile for activity against Ac<subscript>2</subscript>- l-Lys- d-Ala- d-Ala ( k<subscript>cat</subscript>/ K<subscript>m</subscript>) was bell-shaped, with p K<subscript>a</subscript> values at 6.9 and 10.1. Comparison of the enzymatic properties of NG PBP 4 with other dd-carboxypeptidases highlights both similarities and differences within these enzymes, and suggests the possibility of common mechanistic roles for the two highly conserved active site lysines in Class A and C low molecular mass PBPs. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00142956
- Volume :
- 271
- Issue :
- 1
- Database :
- Complementary Index
- Journal :
- European Journal of Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11723896
- Full Text :
- https://doi.org/10.1046/j.1432-1033.2003.03886.x